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Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

ABSTRACT: Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. It is known to be activated by dissociation of the oligomer at heat shock temperatures. We wondered whether phosphorylation regulates its activity at physiological temperatures. In Hsp26, 9 phosphorylation sites which are located in different structural elements are known. Our analysis of phospho-mimetic mutations showed that phosphorylation activates Hsp26 at permissive temperature. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, the C-terminal extension, which also links subunits within the oligomer, contains several phosphorylation sites. The introduction of negative charges at these strategic positions makes regions in the N-terminal domain accessible for the binding of substrate proteins. Relieving the intrinsic inhibition via different phosphorylation sites allows the fine-tuning of chaperone activity in response to proteotoxic stresses independent of heat activation. The described weakening of domain interactions within and between subunits could be a general regulation principle of sHsps.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Moritz Mühlhofer

LAB HEAD: Johannes Buchner

PROVIDER: PXD025314 | Pride | 2021-12-17

REPOSITORIES: pride

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			Action	DRS
	20210329_MM_CL36.kojak.txt	Txt
	20210329_MM_CL36.raw	Raw
	20210329_MM_CL37.kojak.txt	Txt
	20210329_MM_CL37.raw	Raw
	20210329_MM_CL38.kojak.txt	Txt

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The permanently chaperone-active small heat shock protein Hsp17 from C. elegans exhibits topological separation of its N-terminal regions

Project description:Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind non-native proteins in an ATP-independent manner. C. elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under non-stress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

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